Person:
Cruz Rodríguez, Antonio

First Name

Antonio

Last Name

Cruz Rodríguez

URI

https://hdl.handle.net/20.500.14352/76077

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Ciencias Químicas

Department

Bioquímica y Biología Molecular

Area

Bioquímica y Biología Molecular

Identifiers

Full item page

Search Results

Now showing 1 - 10 of 13

Effects of HIV-1 gp41-Derived Virucidal Peptides on Virus-like Lipid Membranes
(Biophysical Journal, 2017) Carravilla, Pablo; Cruz Rodríguez, Antonio; Martín-Ugarte, Itziar; Oar-Arteta, Itziar R.; Torralba, Johanna; Apellaniz, Beatriz; Pérez-Gil, Jesús; Requejo Isidro, José; Huarte, Nerea; Nieva, José L.
Membrane fusion induced by the envelope glycoprotein enables the intracellular replication of HIV-1; hence, this process constitutes a major target for antiretroviral compounds. It has been proposed that peptides having propensity to interact with membrane interfaces might exert broad antiviral activity against enveloped viruses. To test this hypothesis, in this contribution we have analyzed the antiviral effects of peptides derived from the membrane-proximal external region and the transmembrane domain of the envelope glycoprotein subunit gp41, which display different degrees of interfacial hydrophobicity. Our data support the virucidal activity of a region that combines hydrophobic-at-interface membrane-proximal external region aromatics with hydrophobic residues of the transmembrane domain, and contains the absolutely conserved 679LWYIK/R683 sequence, proposed to embody a ‘‘cholesterol recognition/interaction amino acid consensus’’ motif. We further sought to correlate the antiviral activity of these peptides and their effects on membranes that mimic lipid composition and biophysical properties of the viral envelope. The data revealed that peptides endowed with virucidal activity were membrane active and induced permeabilization and fusion of virus-like lipid vesicles. In addition, they modulated lipid packing and miscibility of laterally segregated liquid domains, two properties that depend on the high cholesterol content of the viral membrane. Thus, the overall experimental evidence is consistent with a pattern of HIV inhibition that involves direct alteration of the physical chemistry of the virus membrane. Furthermore, the sequence-dependent effects observed might guide the development of new virucidal peptides.
Pulmonary surfactant and nanocarriers: toxicity versus combined nanomedical applications
(Biochimica et Biophysica Acta - Biomembranes, 2017) Hidalgo Román, Alberto; Cruz Rodríguez, Antonio; Pérez-Gil, Jesús
Pulmonary surfactant is a membrane-based lipid-protein system essential for the process of breathing, which coats and stabilizes the whole respiratory surface and possesses exceptional biophysical properties. It constitutes the first barrier against the entry of pathogens and harmful particles in the alveolar region, extended through the lungs, but on the other hand, it can offer novel possibilities as a shuttle for the delivery of drugs and nanocarriers. The advances in nanotechnology are opening the doors to new diagnostic and therapeutic avenues, which are not accessible by means of the current approaches. In this context, the pulmonary route is called to become a powerful way of entry for innovative treatments based on nanotechnology. In this review, the anatomy of the respiratory system and its properties for drug entry are first revisited, as well as some current strategies that use the respiratory route for both local and peripheral action. Then, a brief overview is presented on what pulmonary surfactant is, how it works and why it could be used as a drug delivery vehicle. Finally, the review is closed with a description of the development of nanocarriers in the lung context and their interaction with endogenous and clinical pulmonary surfactants.
Towards the molecular mechanism of pulmonary surfactant Protein SP-B: at the crossroad of membrane permeability and interfacial lipid transfer
(American Journal of Molecular Biology, 2020) Martínez Calle, Marta; Parra Ortiz, Elisa; Cruz Rodríguez, Antonio; Olmeda Lozano, Bárbara; Pérez-Gil, Jesús
Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.
Molecular and biophysical mechanisms behind the enhancement of lung surfactant function during controlled therapeutic hypothermia
(Scientific Reports, 2020) Autilio, Chiara; Echaide Torreguitar, Mercedes; Cruz Rodríguez, Antonio; Hidalgo, A.; Da Silva, E.; De Luca, Daniele; Sørli, Jorid B.; Pérez-Gil, Jesús
Therapeutic hypothermia (TH) enhances pulmonary surfactant performance in vivo by molecular mechanisms still unknown. Here, the interfacial structure and the composition of lung surfactant flms have been analysed in vitro under TH as well as the molecular basis of its improved performance both under physiological and inhibitory conditions. The biophysical activity of a purifed porcine surfactant was tested under slow and breathing-like dynamics by constrained drop surfactometry (CDS) and in the captive bubble surfactometer (CBS) at both 33 and 37 °C. Additionally, the temperaturedependent surfactant activity was also analysed upon inhibition by plasma and subsequent restoration by further surfactant supplementation. Interfacial performance was correlated with lateral structure and lipid composition of flms made of native surfactant. Lipid/protein mixtures designed as models to mimic diferent surfactant contexts were also studied. The capability of surfactant to drastically reduce surface tension was enhanced at 33 °C. Larger DPPC-enriched domains and lower percentages of less active lipids were detected in surfactant flms exposed to TH-like conditions. Surfactant resistance to plasma inhibition was boosted and restoration therapies were more efective at 33 °C. This may explain the improved respiratory outcomes observed in cooled patients with acute respiratory distress syndrome and opens new opportunities in the treatment of acute lung injury.
Pulmonary surfactant and drug delivery: Vehiculization of a tryptophan-tagged antimicrobial peptide over the air-liquid interfacial highway
(European Journal of Pharmaceutics and Biopharmaceutics, 2022) García-Mouton, Cristina; Parra Ortiz, Elisa; Malmsten, Martin; Cruz Rodríguez, Antonio; Pérez Gil, Jesús
This work evaluates interaction of pulmonary surfactant (PS) and antimicrobial peptides (AMPs) in order to investigate (i) if PS can be used to transport AMPs, and (ii) to what extent PS interferes with AMP function and vice versa. This, in turn, is motivated by a need to find new strategies to treat bacterial infections in the airways. Low respiratory tract infections (LRTIs) are a leading cause of illness and death worldwide that, together with the problem of multidrug-resistant (MDR) bacteria, bring to light the necessity of developing effective therapies that ensure high bioavailability of the drug at the site of infection and display a potent antimicrobial effect. Here, we propose the combination of AMPs with PS to improve their delivery, exemplified for the hydrophobically endtagged AMP, GRR10W4 (GRRPRPRPRPWWWW-NH2), with previously demonstrated potent antimicrobial activity against a broad spectrum of bacteria under various conditions. Experiments using model systems emulating the respiratory interface and an operating alveolus, based on surface balances and bubble surfactometry, served to demonstrate that a fluorescently labelled version of GRR10W4 (GRR10W4-F), was able to interact and insert into PS membranes without affecting its biophysical function. Therefore, vehiculization of the peptide along air–liquid interfaces was enabled, even for interfaces previously occupied by surfactants layers. Furthermore, breathing-like compression-expansion dynamics promoted the interfacial release of GRR10W4-F after its delivery, which could further allow the peptide to perform its antimicrobial function. PS/GRR10W4-F formulations displayed greater antimicrobial effects and reduced toxicity on cultured airway epithelial cells compared to that of the peptide alone. Taken together, these results open the door to the development of novel delivery strategies for AMPs in order to increase the bioavailability of these molecules at the infection site via inhaled therapies.
In Vitro functional and structural characterization of a synthetic clinical pulmonary surfactant with enhanced resistance to inhibition
(Scientific Reports, 2020) Echaide Torreguitar, Mercedes; Autilio, Chiara; López-Rodríguez, Elena; Cruz Rodríguez, Antonio; Pérez-Gil, Jesús
CHF5633 is a novel synthetic clinical pulmonary surfactant preparation composed by two phospholipid species, dipalmitoyl phosphatidylcholine (DPPC) and palmitoyloleoyl phosphatidylglycerol (POPG), and synthetic analogues of the hydrophobic surfactant proteins SP-B and SP-C. In this study, the interfacial properties of CHF5633 in the absence and in the presence of inhibitory serum proteins have been assessed in comparison with a native surfactant purifed from porcine lungs and with poractant alpha, a widely used clinical surfactant preparation. The study of the spreading properties of CHF5633 in a Wilhelmy balance, its ability to adsorb and accumulate at air-liquid interfaces as revealed by a multiwell fuorescence assay, and its dynamic behavior under breathing-like compression-expansion cycling in a Captive Bubble Surfactometer (CBS), all revealed that CHF5633 exhibits a good behavior to reduce and sustain surface tensions to values below 5 mN/m. CHF5633 shows somehow slower initial interfacial adsorption than native surfactant or poractant alpha, but a better resistance to inhibition by serum proteins than the animal-derived clinical surfactant, comparable to that of the full native surfactant complex. Interfacial CHF5633 flms formed in a Langmuir-Blodgett balance coupled with epifuorescence microscopy revealed similar propensity to segregate condensed lipid domains under compression than flms made by native porcine surfactant or poractant alpha. This ability of CHF5633 to segregate condensed lipid phases can be related with a marked thermotropic transition from ordered to disordered membrane phases as exhibited by diferential scanning calorimetry (DSC) of CHF5633 suspensions, occurring at similar temperatures but with higher associated enthalpy than that shown by poractant alpha. The good interfacial behavior of CHF5633 tested under physiologically meaningful conditions in vitro and its higher resistance to inactivation by serum proteins, together with its standardized and well-defned composition, makes it a particularly useful therapeutic preparation to be applied in situations associated with lung infammation and edema, alone or in combined strategies to exploit surfactant-facilitated drug delivery.
Functional organization of the HIV lipid envelope
(Scientific Reports, 2016) Huarte, Nerea; Carravilla, Pablo; Cruz Rodríguez, Antonio; Lorizate, Maier; Nieto-Garay, Jon A.; Kräusslich, Hans-Georg; Pérez-Gil, Jesús; Requejo Isidro, José; Nieva, José L.
The chemical composition of the human immunodeficiency virus type 1 (HIV-1) membrane is critical for fusion and entry into target cells, suggesting that preservation of a functional lipid bilayer organization may be required for efficient infection. HIV-1 acquires its envelope from the host cell plasma membrane at sites enriched in raft-type lipids. Furthermore, infectious particles display aminophospholipids on their surface, indicative of dissipation of the inter-leaflet lipid asymmetry metabolically generated at cellular membranes. By combining two-photon excited Laurdan fluorescence imaging and atomic force microscopy, we have obtained unprecedented insights into the phase state of membranes reconstituted from viral lipids (i.e., extracted from infectious HIV-1 particles), established the role played by the different specimens in the mixtures, and characterized the effects of membrane-active virucidal agents on membrane organization. In determining the molecular basis underlying lipid packing and lateral heterogeneity of the HIV-1 membrane, our results may help develop compounds with antiviral activity acting by perturbing the functional organization of the lipid envelope.
The lord of the Lungs: the essential role of pulmonary surfactant upon inhalation of nanoparticles
(European Journal of Pharmaceutics and Biopharmaceutics, 2019) García-Mouton, Cristina; Hidalgo Román, Alberto; Cruz Rodríguez, Antonio; Pérez-Gil, Jesús
The rapid development of nanotechnology is opening a huge world of promising possibilities in healthcare, but this is also increasing the necessity to study the potential risk of nanoparticles on public health and the environment. Since the main route for airborne particles to enter into our organism is through the lungs, it has become essential to prove that the nanoparticles generated by human activities do not compromise the respiratory function. This review explains the key role of pulmonary surfactant to sustain the normal function of breathing, as well as the stability and immunity of lungs. Particular emphasis is made on the importance of analysing the features of nanoparticles, defining their interactions with surfactant and unravelling the mutual effects. The implication of the nanoparticle-surfactant interaction on the function and fate of both structures is described, as well as the main in vitro methodologies used to evaluate this interaction. Finally, the incorporation of pulmonary surfactant in appropriate in vitro models is used in order to obtain an extensive understanding of how nanoparticles may act in the context of the lung. The main goal of this review is to offer a general view on inhaled nanoparticles and their effects on the structure and function of lungs derived from their interaction with the pulmonary surfactant system.
The highly packed and dehydrated structure of preformed unexposed human pulmonary surfactant isolated from amniotic fluid
(American Journal of Physiology - Lung Cellular and Molecular Physiology (AJP - Lung Cellular and Molecular Physiology), 2022) Castillo Sánchez, José Carlos; Roldán, Nuria; García Álvarez, Begoña; Batllori, Emma; Galindo Izquierdo, Alberto; Cruz Rodríguez, Antonio; Pérez Gil, Jesús
By coating the alveolar air-liquid interface, lung surfactant overwhelms surface tension forces that, otherwise, would hinder the lifetime effort of breathing. Years of research have provided a picture of how highly hydrophobic and specialized proteins in surfactant promote rapid and efficient formation of phospholipid-based complex three-dimensional films at the respiratory surface, highly stable under the demanding breathing mechanics. However, recent evidence suggests that the structure and performance of surfactant typically isolated from bronchoalveolar lung lavages may be far from that of nascent, still unused, surfactant as freshly secreted by type II pneumocytes into the alveolar airspaces. In the present work, we report the isolation of lung surfactant from human amniotic fluid (amniotic fluid surfactant, AFS) and a detailed description of its composition, structure, and surface activity in comparison to a natural surfactant (NS) purified from porcine bronchoalveolar lavages. We observe that the lipid/ protein complexes in AFS exhibit a substantially higher lipid packing and dehydration than in NS. AFS shows melting transitions at higher temperatures than NS and a conspicuous presence of nonlamellar phases. The surface activity of AFS is not only comparable with that of NS under physiologically meaningful conditions but displays significantly higher resistance to inhibition by serum or meconium, agents that inactivate surfactant in the context of severe respiratory pathologies. We propose that AFS may be the optimal model to study the molecular mechanisms sustaining pulmonary surfactant performance in health and disease, and the reference material to develop improved therapeutic surfactant preparations to treat yet unresolved respiratory pathologies.
Biophysical and biological impact on the structure and IgE-binding of the interaction of the olive pollen allergen Ole e 7 with lipids
(Biochimica et Biophysica Acta (BBA) - Biomembranes, 2020) Oeo-Santos, Carmen; López Rodríguez, Juan Carlos; García Mouton, Cristina; San Segundo Acosta, Pablo; Jurado, Aurora; Moreno-Aguilar, Carmen; García Álvarez, María Begoña; Pérez Gil, Jesús; Villalba Díaz, María Teresa; Barderas Manchado, Rodrigo; Cruz Rodríguez, Antonio
Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways.